The proposed pilot project intends to develop a quantitative mass spectrometric method for studying multisite phosphorylation in a detailed and controlled manner, and to apply that method to the multisite phosphorylation of cyclin-dependent kinase substrates. The detailed study of multisite phosphorylation has been hampered by the lack of techniques capable of quantitatively analyzing phosphorylation dynamics under well-defined conditions. Here, stable isotope-labeling coupled to mass spectrometry will be applied to in vitro phosphorylation reactions between the budding yeast cyclin-dependent kinase Cdk1 and a number of substrate molecules. Stable isotope-labeling/mass spectrometry is a well-established technique with the appropriate sensitivity, dynamic range and precision for making the measurements that will be required to accurately quantify protein phosphorylation in a time-resolved, site-specific manner. Published and unpublished preliminary studies have been performed to show that Cdk1 can be isolated from yeast cells in an active form, and can be used for in vitro phosphorylation reactions. This proposal aims to understand multisite Cdk1 phosphorylation as a biophysical system, and therefore requires the precise control afforded by in vitro kinetic studies. The in vitro approach is preferred because hidden variables are invariably present in the complicated in vivo cellular milieu, such as cross-talk between related enzymes and the presence of diverse protein populations within a single culture. Specifically, this proposal aims to determine the processivity or distributivity of Cdk1 phosphorylation;and the order and dependence, if any, of sites are phosphorylated. Furthermore, Cdk1 has 9 potential in vivo cyclin binding partner/activators present at different points in cell cycle that have partially overlapping specificity and function. Results of these multisite phosphorylation studies may shed insight into differences between the cyclins and modes of cyclin specificity. A major part of the pilot project will also be the professional development of the PI through collaboration with a highly successful mentor, research dissemination, establishment of collaborations, and ultimately, the acquisition of future funding. RELEVANCE TO PUBLIC HEALTH: This proposal aims to utilize leading-edge analytical technology to understand the activity of cyclin dependent kinases, a main player in cell division. Knowledge gained from the proposed study on the model eukaryote S. cerevisiae will have value in elucidating principles that also human biology, particularly as it relates to regulation of cell proliferation and cancer. The innovative in vitro phosphorylation/quantitative mass spectrometry approach proposed also lends itself readily to studying other multisite enzyme systems, such as the glycogen synthase kinases, and casein kinases-proteins that are intimately involved in processes such as metabolic regulation, inflammation, and Alzheimer's disease and other neurological disorders.